The Effect of Formalin Fixation on Resection Margins in Colorectal Cancer.

Resection margins in colorectal cancer carry clinical significance with regard to disease recurrence risk and selection for multimodal adjuvant therapy, especially with circumferential resection margins in rectal cancer. Colorectal cancer specimens are routinely fixed in formalin, which results in specimen and tumor-free margin shrinkage. However, the effects of shrinkage have not traditionally been taken into account when analyzing tumor-free margins. In this prospective study, 46 colorectal cancer specimens were measured in the fresh state and subsequently after formalin fixation for total specimen length, distal resection margin, and radial margin (circumferential resection margin for rectal cancer). The mean reduction after formalin fixation was 17.48 mm (14.7%) for distal resection margin and 1.20 mm (10.5%) for radial margin. For rectal cancer, circumferential resection margin reduction was 0.88 mm (11.8%); this was not affected by neoadjuvant chemoradiotherapy. Duration of formalin fixation did not significantly affect the extent of margin shrinkage. This is the first study to evaluate the effect of formalin fixation on radial resection margins, specifically as it relates to rectal cancer, and it demonstrates that shrinkage from formalin fixation should be a consideration in decision-making where the magnitude of tumor-free margins is small.

Dysfunction of ventrolateral prefrontal cortex underlying social anxiety disorder: A multi-channel NIRS study.

Social anxiety disorder (SAD) is characterized by strong fear and anxiety during social interactions. Although ventrolateral prefrontal cortex (VLPFC) activity in response to emotional stimuli is related to pathological anxiety, little is known about the relationship between VLPFC activity and social anxiety. This study aimed to investigate whether VLPFC activity was involved in SAD and whether VLPFC activity was related to the level of social anxiety. Twenty-four drug-naïve patients with SAD and 35 healthy controls underwent near-infrared spectroscopy (NIRS) scanning while performing a verbal fluency task (VFT). Results indicated that, compared to the healthy controls, the SAD patients exhibited smaller changes of oxygenated hemoglobin (oxy-Hb) concentrations in the VLPFC during the VFT. Furthermore, the right VLPFC activation was negatively correlated with social avoidance. In contrast to the latter, the healthy controls exhibited a positive correlation between changes of oxy-Hb concentrations in the bilateral VLPFC and social fear. Our findings provide evidence for VLPFC dysfunction in SAD, and indicate that the VLPFC dysfunction may contribute to the difference between normal and abnormal social anxiety.

Improving patient selection for 18F-FDG PET scanning in the staging of gastric cancer.

UNLABELLED: Standard pretreatment staging for gastric cancer includes CT of the chest, abdomen, and pelvis; gastroscopy; and laparoscopy. Although (18)F-PET combined with CT has proven to be a useful staging tool in many cancers, some gastric cancers are not (18)F-FDG-avid and its clinical value is still debatable. METHODS: Gastric cancer patients who underwent staging (18)F-FDG PET scans from 2002 to 2013 at the Peter MacCallum Cancer Center were retrospectively analyzed, and a systematic review was also conducted using PubMed between 2000 to March 2014 to investigate clinicopathologic parameters associated with (18)F-FDG avidity. A pretreatment PET scoring system was developed from predictors of (18)F-FDG avidity. RESULTS: Both the retrospective analysis of the patients and the systematic literature review showed similar significant predictors of (18)F-FDG avidity, including large tumor size, non-signet ring cell carcinoma type, and glucose transporter 1-positive expression on immunohistochemistry. A PET scoring system was developed from these clinicopathologic parameters that allowed (18)F-FDG-avid tumors to be detected with a sensitivity of 85% and a specificity of 71%. CONCLUSION: A pretreatment PET scoring system can assist in the selection of patients with gastric adenocarcinoma when staging (18)F-FDG PET is being considered.

A systematic review and meta-analysis of gastric cancer treatment in patients with positive peritoneal cytology.

Gastric cancer patients with positive peritoneal cytology as the only marker of metastatic disease have poor prognoses. There is no universal consensus on the most appropriate treatment regimen for this particular patient group. We reviewed and analyzed published data to determine the optimal treatment regimen for patients with peritoneal cytology-positive gastric adenocarcinomas. Six electronic databases were explored [PubMed, Cochrane (Systematic Reviews and Controlled Trials), PROSPERO, DARE, and EMBASE]. The primary outcome was overall survival with secondary outcomes including patterns of recurrence and treatment-related morbidity. Six studies were included for data extraction. There was no significant heterogeneity between studies. The use of S1 monotherapy was associated with a significant survival benefit (HR 0.48; 95% CI 0.32-0.70; p = 0.0002). Intraoperative intraperitoneal chemotherapy (IIPC) with adjuvant chemotherapy showed a trend toward improvement in overall survival (HR 0.70; 9 % CI 0.47-1.04; p = 0.08). A recent randomized controlled trial examining extensive intraperitoneal lavage (EIPL) with IIPC showed a significant improvement in overall survival (5-year overall survival, 43.8% for EIPL-IPC group compared with 4.6% for IPC group). However, these promising results need to be validated in larger prospective randomized trials.

Calpain 2 activity increases at the time of implantation in rat uterine luminal epithelial cells and administration of calpain inhibitor significantly reduces implantation sites.

The present study investigated the role of calpain 2 in rat uterine luminal epithelial cells during early pregnancy. Calpain 2 is an intracellular calcium-dependent proteolytic enzyme which cleaves numerous focal adhesion proteins. Calpain 2 was concentrated along the basal cell surface of uterine luminal epithelial cells at the predicted site of focal adhesions on day 1 of pregnancy and remained unchanged at the time of implantation as observed by immunofluorescence microscopy. However, Western blotting analysis showed a marked increase in the active form and a significant decrease in the latent form of calpain 2 at the time of implantation. The increase in calpain 2 activity coincides with the disassembly of focal adhesion proteins, talin, paxillin, integrin ß1 and ß3 from the site of focal adhesions. Intraperitoneal injection of calpain inhibitor, calpain inhibitor l (ALLN), significantly reduced the number of implantation sites, implying that calpain 2 plays an important role in implantation. The present study suggests a role for calpain 2 in the disassembly of focal adhesions, which has been previously shown to play a key role in uterine receptivity for implantation.

Extracellular matrix proteins secreted from both the endometrium and the embryo are required for attachment: a study using a co-culture model of rat blastocysts and Ishikawa cells.

Integrins are expressed in a highly regulated manner at the maternal-fetal interface during implantation. However, the significance of extracellular matrix (ECM) ligands during the integrin-mediated embryo attachment to the endometrium is not fully understood. Thus, the distribution of fibronectin in the rat uterus and blastocyst was studied at the time of implantation. Fibronectin was absent in the uterine luminal epithelial cells but was intensely expressed in the trophoblast cells and the inner cell mass suggesting that fibronectin secreted from the blastocyst may be a possible bridging ligand for the integrins expressed at the maternal-fetal interface. An Arg-Gly-Asp (RGD) peptide was used to block the RGD recognition sites on integrins, and the effect on rat blastocyst attachment to Ishikawa cells was examined. There was a significant reduction in blastocyst attachment when either the blastocysts or the Ishikawa cells were pre-incubated with the RGD-blocking peptide. Thus, successful attachment of the embryo to the endometrium requires the interaction of integrins on both the endometrium and the blastocyst with the RGD sequence of ECM ligands, such as fibronectin. Pre-treatment of both blastocysts and Ishikawa cells with the RGD peptide also inhibited blastocyst attachment, but not completely, suggesting that ECM bridging ligands that do not contain the RGD sequence are also involved in embryo attachment.

Uterine epithelial cells: Serving two masters.

Uterine epithelial cells are unique cells in that they are both epithelial in the typical barrier sense but in many mammalian species, they characteristically allow the blastocyst to penetrate them from the apical surface. Here we examine how these cells subserve both functions and in particular we synthesize recent evidence on focal adhesions and how these membrane structures contribute to uterine receptivity for blastocyst implantation. Focal adhesions emerge as a dynamic new player in the 'plasma membrane transformation' of early pregnancy and uterine receptivity in that they disassemble at the time of implantation in common with many other structures on the basolateral plasma membrane of these cells.

Focal adhesion kinase localizes to sites of cell-to-cell contact in vivo and increases apically in rat uterine luminal epithelium and the blastocyst at the time of implantation.

Focal adhesions play an important role in promoting embryo invasion; in particular, focal adhesions disassemble at the time of implantation in the rat, facilitating the detachment of the uterine luminal epithelium to allow the embryo to invade the endometrium. This study investigated focal adhesion protein, focal adhesion kinase (FAK) in the rat uterine luminal, and glandular epithelial cells to understand the dynamics of focal adhesions during early pregnancy. FAK undergoes extensive distributional change during early pregnancy, and surprisingly, FAK was not localized at the site of focal adhesions, instead being localized to the site of cell-to-cell contact and colocalizing with ZO-1 on day 1 of pregnancy. At the time of implantation, FAK increases in the apical region of the uterine luminal epithelial cells which was regulated by progesterone. Using an in vitro co-culture model of rat blastocysts attached to Ishikawa cells, FAK was present apically both in the rat blastocyst and the Ishikawa cells, suggesting a role in attachment andin mediating signal transduction between these two genetically different cell types.

Integrin ß3 in rat blastocysts and epithelial cells is essential for implantation in vitro: studies with Ishikawa cells and small interfering RNA transfection.

BACKGROUND: Integrins are involved in the process of embryo-endometrium interaction during implantation. We investigated the localization of integrin ß3 in the rat blastocyst and Ishikawa cells using an in vitro co-culture model of implantation. METHODS: Zona pellucida-free rat blastocysts were co-cultured with the Ishikawa cells (endometrial adenocarcinoma cell line) to observe the attachment between the embryo and endometrium. Immunofluorescence staining was used to investigate the localization of integrin ß3 in rat embryos at different stages of development (each n= 3 embryos) and at the embryo/endometrium interface, observed by confocal microscopy. The Ishikawa cells were transfected with integrin ß3 small interfering RNA (siRNA) for 48 h and then co-cultured with Day 5 rat blastocysts to observe the effect on attachment. RESULTS: Integrin ß3 staining in the rat embryos increased at the blastocyst stage being highly concentrated in the cytoplasm of trophoblast cells (n= 9 embryos). Integrin ß3 was localized on the apical surface of the Ishikawa cells (n= 3 experiments). However, integrin ß3 relocated to the apical membrane of trophoblast cells in response to attachment to Ishikawa cells (n= 6 embryos). Moreover, when Ishikawa cells were transfected with integrin ß3 siRNA, blastocyst attachment was significantly reduced compared with those transfected with control siRNA (16.7 versus 92.3%, respectively, P< 0.05). CONCLUSIONS: Integrin ß3, localized apically in the blastocyst and the Ishikawa cells, is important during initial attachment of the blastocyst to endometrial cells. This study provides further evidence of the importance of integrins during implantation and may aid in elucidating the molecular mechanism of implantation failure and infertility in women.

ß(1) and ß(3) integrins disassemble from basal focal adhesions and ß(3) integrin is later localised to the apical plasma membrane of rat uterine luminal epithelial cells at the time of implantation.

The present study investigated the expression of integrin subunits that are known to be associated with focal adhesions, namely ß(1) and ß(3) integrins in rat uterine luminal epithelial cells during early pregnancy. The ß(1) and ß(3) integrins were concentrated along the basal cell surface and were colocalised and structurally interacted with talin, a principal focal adhesion protein, on Day 1 of pregnancy. At the time of implantation, ß(1) and ß(3) integrins disassembled from the site of focal adhesions, facilitating the removal of uterine luminal epithelial cells for embryo invasion. Also at this time, ß(3) integrin markedly increased along the apical membrane, suggesting a role in embryo attachment. This distributional change in ß(1) and ß(3) integrins seen at the time of implantation was predominantly under the influence of progesterone. Taken together, ß(1) and ß(3) integrin disassembly from focal adhesions and the increase in ß(3) integrin apically are key components of hormonally regulated endometrial receptivity.

ICAM1 and fibrinogen-γ are increased in uterine epithelial cells at the time of implantation in rats.

Uterine epithelial cells transform into a receptive state to adhere to an implanting blastocyst. Part of this transformation includes the apical concentration of cell adhesion molecules at the time of implantation. This study, for the first time, investigates the expression of ICAM1 and fibrinogen-γ (FGG) in uterine epithelial cells during normal pregnancy, pseudopregnancy and in hormone-treated rats. An increase (P < 0.05) in ICAM1 was seen at the apical membrane of uterine epithelial cells at the time of implantation compared with day 1 of pregnancy. ICAM1 was also increased (P < 0.05) on day 6 of pseudopregnancy as well as in ovariectomized rats treated with progesterone plus oestrogen. These results show that ICAM1 up-regulation at the time of implantation is under the control of progesterone, and is not dependent on cytokine release from the blastocyst or in semen. FGG dimerization increased (P < 0.05) on day 6 of pregnancy compared with day 1, and was not up-regulated in day 6 pseudopregnant animals, suggesting this increase is dependent on a developing blastocyst. The presence of ICAM1 and FGG in the uterine epithelium at the time of implantation in the rat is similar to that seen in lymphocyte-endothelium adhesion, and we suggest a similar mechanism in embryo-uterine epithelium adhesion is utilized.

Ovarian hormones regulate expression of the focal adhesion proteins, talin and paxillin, in rat uterine luminal but not glandular epithelial cells.

During early pregnancy in the rat, focal adhesions disassemble in uterine luminal epithelial cells at the time of implantation to facilitate their removal so that the implanting blastocyst can invade into the underlying endometrial decidual cells. This study investigated the effect of ovarian hormones on the distribution and protein expression of two focal adhesion proteins, talin and paxillin, in rat uterine luminal and glandular epithelial cells under various hormone regimes. Talin and paxillin showed a major distributional change between different hormone regimes. Talin and paxillin were highly concentrated along the basal cell surface of uterine luminal epithelial cells in response to oestrogen treatment. However, this prominent staining of talin and paxillin was absent and also a corresponding reduction of paxillin expression was demonstrated in response to progesterone alone or progesterone in combination with oestrogen, which is also observed at the time of implantation. In contrast, the distribution of talin and paxillin in uterine glandular epithelial cells was localised on the basal cell surface and remained unchanged in all hormone regimes. Thus, not all focal adhesions are hormonally dependent in the rat uterus; however, the dynamics of focal adhesion in uterine luminal epithelial cells is tightly regulated by ovarian hormones. In particular, focal adhesion disassembly in uterine luminal epithelial cells, a key component to establish successful implantation, is predominantly under the influence of progesterone.

Focal adhesions disassemble during early pregnancy in rat uterine epithelial cells.

During early pregnancy in rodents, invasion of the blastocyst into the endometrial decidual cells is accompanied by the removal of uterine epithelial cells around the implantation sites. The present study investigated the distribution and expression of two focal adhesion proteins, namely talin and paxillin, in rat uterine epithelial cells during early pregnancy and their role in the loss of these cells at the time of implantation. A major distributional change of talin and paxillin was demonstrated in uterine epithelial cells during early pregnancy. From a highly concentrated expression along the basal cell surface on Day 1 of pregnancy, talin and paxillin were lost from the basal cell surface at the time of implantation. There was also a corresponding statistically significant decrease in paxillin seen through western blotting analysis. Together, these observations suggest that uterine epithelial cells are less adherent to the underlying basal lamina due to the disassembly of talin and paxillin from focal adhesions, facilitating removal of these cells at the time of implantation. This phenomenon was restricted to the period of receptivity because talin and paxillin reappeared along the basal cell surface soon after implantation.